Previously, we constructed recombinant vaccinia viruses that expressed individually the hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of human parainfluenza virus type 3 (PIV3). Intradermal immunization of rodents and non-human primates induced high levels of protective immunity and demonstrated the feasibility of a subunit vaccine for PIV3. In continuation of this work, we are constructing analogous recombinant vaccinia viruses using parental strains (Wyeth and Lister) that are suitable for evaluating immunogenicity in humans. Also, recombinant adenoviruses that will express individually the HN and F proteins are under construction. The intracellular synthesis and maturation of the HN protein was investigated by the expression of cDNAs that had been engineered to encode N-terminally truncated HN proteins. The truncated HN proteins contained progressively larger deletions of the N-terminal cytoplasmic and transmembrane domains. The results indicated that the folding of the HN ectodomain into a biologically active and conformationally authentic form occurs to completion in the rough endoplasmic reticulum. However, the finding that two biologically- active HN mutants remained localized primarily in the rough endoplasmic reticulum indicated that correct folding was not the sole prerequisite for movement through the exocytotic pathway. Finally, the glycosylation of the HN protein (which contains only N-linked sugars) and the respiratory syncytial virus G protein (which contains both N-linked and O-linked sugars) was compared. By cDNA mutagenesis and expression, chimeric proteins were expressed that consisted of the N-terminal cytoplasmic and membrane domains of each protein fused to the ectodomain of the other. This study showed that the processing and maturation of each ectodomain was independent of the origin of the cytoplasmic and membrane domains.